Armstrong K M, Quigley E P, Quigley P, Crumrine D S, Cukierman S
Department of Physiology, Loyola University Medical School, Maywood, Illinois 60153, USA.
Biophys J. 2001 Apr;80(4):1810-8. doi: 10.1016/S0006-3495(01)76151-6.
The direct role of the dioxolane group on the gating and single-channel conductance of different stereoisomers of the dioxolane-linked gramicidin A (gA) channels reconstituted in planar lipid bilayers was investigated. Four different covalently linked gA dimers were synthesized. In two of them, the linker was the conventional dioxolane described previously (SS and RR channels). Two gAs were covalently linked with a novel modified dioxolane group containing a retinal attachment (ret-SS and ret-RR gA dimers). These proteins also formed ion channels in lipid bilayers and were selective for monovalent cations. The presence of the bulky and hydrophobic retinal group immobilizes the dioxolane linker in the bilayer core preventing its rotation into the hydrophilic lumen of the pore. In 1 M HCl the gating kinetics of the SS or RR dimers were indistinguishable from their retinal counterparts; the dwell-time distributions of the open and closed states in the SS and ret-SS were basically the same. In particular, the inactivation of the RR was not prevented by the presence of the retinal group. It is concluded that neither the fast closing events in the SS or RR dimers nor the inactivation of the RR are likely to be a functional consequence of the flipping of the dioxolane inside the pore of the channel. On the other hand, the inactivation of the RR dimer was entirely eliminated when alkaline metals (Cs(+) or K(+)) were the permeating cations in the channel. In fact, the open state of the RR channel became extremely stable, and the gating characteristics of both the SS and RR channels were different from what was seen before with permeating protons. As in HCl, the presence of a retinal in the dioxolane linker did not affect the gating behavior of the SS and RR in Cs(+)- or K(+)-containing solutions. Alternative hypotheses concerning the gating of linked gA dimers are discussed.
研究了二氧戊环基团对在平面脂质双层中重构的二氧戊环连接的短杆菌肽A（gA）通道不同立体异构体的门控和单通道电导的直接作用。合成了四种不同的共价连接的gA二聚体。其中两种，连接基团是先前描述的传统二氧戊环（SS和RR通道）。另外两种gA通过含有视黄醛附着的新型修饰二氧戊环基团共价连接（ret-SS和ret-RR gA二聚体）。这些蛋白质也在脂质双层中形成离子通道，并且对单价阳离子具有选择性。庞大且疏水的视黄醛基团的存在使二氧戊环连接基团固定在双层核心中，阻止其旋转到孔的亲水性内腔中。在1 M HCl中，SS或RR二聚体的门控动力学与其含有视黄醛的对应物没有区别；SS和ret-SS中开放和关闭状态的驻留时间分布基本相同。特别是，视黄醛基团的存在并没有阻止RR的失活。得出的结论是，SS或RR二聚体中的快速关闭事件以及RR的失活都不太可能是通道孔内二氧戊环翻转的功能后果。另一方面，当碱金属（Cs(+)或K(+)）是通道中的渗透阳离子时，RR二聚体的失活被完全消除。事实上，RR通道的开放状态变得极其稳定，并且SS和RR通道的门控特性与之前渗透质子时不同。与在HCl中一样，二氧戊环连接基团中视黄醛的存在并不影响SS和RR在含Cs(+)或K(+)溶液中的门控行为。讨论了关于连接的gA二聚体门控的其他假设。
